Numbers of muscle nuclei and myosatellite cell nuclei in red and white axial muscle during growth of the carp (Cyprinus carpio)

We determined the percentages of muscle fibie nuclei and satellite nuclei over a growth range of carp ( Cyprinus carpio ), as the increase in the number of muscle fibre nuclei is an important aspect of the increase in muscle mass, and myosatellite cells are believed to be the source of new muscle fibre nuclei. In white as well as in red axial muscle the percentage of the nuclei present in muscle that are muscle nuclei (muscle fibre nuclei+myosatellite nuclei) remained constant during growth (54 and 32% respectively). The difference in the percentage of non-muscle nuclei between white and red axial muscle is mainly caused by the higher content of endothelial nuclei in red axial muscle.
In white axial muscle the DNA/protein ratio (nucleus/sarcoplasm ratio) decreased between 3 and 15 cm S.l. In red axial muscle we found a continuous decrease in DNA/protein ratio over the entire investigated size range (3–50 cm s.l.). This may be related to a longer occurrence of hyperplasia in red than in white axial muscle.
In both fibre types the percentage of muscle nuclei being myosatellite nuclei decreased with increasing length, In white axial muscle it decreased from about 5% in carp of 5 cm s.l. to less than 1% in carp of 20 cm S.L.; for red muscle these values were 11 and 3% respectively.
For white axial muscle we calculated that, especially in larger fish, the myosatellite ceils alone cannot account for the increase in the number of muscle fibre nuclei during growth. The percentage of proliferating nuclei in muscle tissue, measured by the uptake of 5-bromo-2'-deoxy-uridine, is high enough to account for the total increase in nuclei. So indirect evidence is available that another cell type present in the muscle tissue may also be involved in the formation of additional muscle fibre nuclei.     

Influence of fish size on proliferation and differentiation of cultured myosatellite cells of white axial muscle of carp (Cyprinus carpio L.)

Abstract. The in vitro proliferation [uptake of 5-bromo-2'-deoxyuridine (BrdU)] and the degree of differentiation (presence of desmin) of myosatellite cells isolated from white axial muscle of carp between 3 cm and 27 cm standard length (SL) were examined 17 h after isolation. The fraction of the myosatellite cells that were both desmin positive and BrdU positive never exceeded 2% of the total number of isolated myosatellite cells, irrespective of the standard length of the donor(s). This indicates that, for carp, the temporal relationship between replication and desmin expression of myosatellite cells is different from that described for myogenic cells of mammals and birds. The percentage of BrdU positive myosatellite cells was significantly correlated with standard length: it increased from 10% for carp of about 5 cm SL to 40–50% for carp between 20 cm and 27 cm SL. The percentage of desmin positive myosatellite cells was about 50–60%; it was not significantly correlated with standard length. The percentage of myosatellite cells that were both BrdU negative and desmin negative showed a stepwise difference in this percentage with increasing length. Fish smaller than 10 cm SL, had more of these cells (10–40%), than larger fish (which had 0–12%). So, apparently the composition of the myosatellite cell population changes during growth. The low percentage of proliferating cells, and the relatively high percentage of differentiated (desmin positive) myosatellite cells obtained from 3–6 cm large carp, suggests that, in these small fish, muscle growth strongly depends on the use of a pool of myogenic cells that has been formed at an earlier stage of their development.     

Numbers of myosatellite cells in white axial muscle of growing fish: Cyprinus carpio L. (Teleostei).

By means of transmission electron microscopy (TEM), the percentage of myosatellite cells was shown to decrease from about 6% in carp of 5 cm standard length (SL) to less than 1% in carp larger than 18 cm SL. The ratio between muscle nuclei and non muscle nuclei remained constant. These TEM data, combined with data on the amount of DNA per gram of muscle tissue and per nucleus, were used to calculate the numbers of myosatellite cells per gram of tissue (TEM-DNA method). Total numbers of myosatellite cells could be calculated from the TEM-DNA data and the calculated amounts of muscle tissue per fish. After a slight initial increase, the total number of myosatellite cells in the white axial muscle of a carp appears to be rather constant during the growth phase of the fish. But the myosatellite cells become more and more diluted over an increasing number of myonuclei with age. In addition, the reliability of two new light microscopic methods for the determination of numbers of myosatellite cells was examined. The percentages of myosatellite cells were determined by counting the numbers of total nuclei and of heterochromatic nuclei situated inside the muscle fibers' basal laminae which were stained using an antibody against laminin. These percentages were not significantly different from those determined with TEM. The yield of myosatellite cells per gram of muscle tissue, isolated with a previously developed dissociation method, showed a direct relation to the number of myosatellite cells calculated to be present in the tissue (TEM-DNA method), but at a 1% level. Both methods are alternative ways to determine numbers of myosatellite cells when it is impossible or difficult to use TEM (e.g., large sample sizes, combination with immunohistochemical methods).     

Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation,recognition and differentiation

Summary We have developed a method for the dissociation and purification of myosatellite cells from white epaxial muscle of carp. The dissociated myosatellite cells were identified by their morphology, their ultrastructure, the formation of multinucleated myotubes containing myofibrils and the immunocytochemical demonstration of desmin. Desmin and 5-bromo-2-deoxyuridine (BrdU) were used to identify terminally differentiated and proliferating myosatellite cells, respectively. The in vitro behavior of myosatellite cells dissociated from carp of 5 cm standard length differed from that described for myosatellite cells of mammals and birds. No substantial proliferation of the myosatellite cells could be observed. Most cells were differentiated (desmin-positive, BrdU-negative) 17 h after plating, regardless of the medium used. This indicates that the investigated white epaxial muscle of carp of 5 cm standard length contains subpopulations of myosatellite cells, arrested at various stages of differentiation.     

Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis

The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).     

The molecular regulation of exercised-induced muscle fibre hypertrophy in the common carp: expression of MyoD, PCNA and components of the calcineurin-signalling pathway

Growth was investigated over 16 d in juvenile common carp (Cyprinus carpio L.) held in either static water (tank rested, TR16) or exercised in a flume at 2.5-3.2 body lengths s-1 for 18 h a day (exercised, E16). Relative to the start of the experiment (TR0), the TR16 group showed a 31% increase in body mass (specific growth rate, 1.57% d-1), whereas there was no net change in the E16 group. There was, however, a significant exercise-induced hypertrophy of slow muscle fibres with average fibre cross-sectional area (FCSA) increasing by 35% in the E16 group, compared with 11% in the TR16 group. In contrast, FCSA of fast muscle fibres increased by 34% in the TR16 group compared to just 18% in the E16 group. The relative concentrations and subcellular localisation of proteins hypothesised to play a role in the regulation of muscle growth were measured. MyoD concentration was similar in the TR0, TR16 and E16 groups in both slow and fast muscle. However, there was a small (5%-10%) but statistically significant increase in nuclear localisation of MyoD in those groups showing a significant increase in FCSA over the time course of the experiment. PCNA concentration was 31% and 12% higher in the TR16 than in either the TR0 or E16 groups for slow and fast muscle, respectively. Exercise resulted in a approximately 10% increase in nuclear factor of T-cells (NFAT2) concentration in slow muscle but no change in NFAT2 localisation. Calcineurin B concentration was similar in tank rested and exercised groups. The results do not support a major role for the calcineurin-signalling pathway in the regulation of muscle hypertrophy in the common carp.     

Muscle fibre growth dynamics in diploid and triploid rainbow trout

The effect of triploidy on muscle fibre growth was determined by comparing hyperplasia and hypertrophy of white muscle fibres in all-female, diploid and triploid rainbow trout Oncorhynchus mykiss (100–400 mm total length). Conventional morphometry and protein and DNA concentrations were used to assess muscle fibre hyperplasia and hypertrophy in white muscle samples derived from an anterio-dorsal location. Muscle fibre distributions were significantly different between triploids and diploids in trout <300 mm. The proportion of fibres <20 μm was higher in diploids than in triploids and the proportion of fibres in the 20–40 μm category was higher in triploids than in diploids. This indicates that the hyperplastic fibres of triploids are larger than those of diploids. Larger hyperplastic fibres in triploids are probably due to the combined effect of increased nuclear size in triploids and the relatively high nucleus: cell ratio observed in small muscle fibres. These larger fibres may be less favourable to cellular metabolic exchange because of their smaller surface area to volume ratios, and perhaps account for reduced viability and growth observed in triploids during early life stages. On the other hand, the lack of difference in the distribution of fibres <20 μm between diploids and triploids at larger body size ranges (301–400 mm) imply that triploid trout may have higher rates of new fibre recruitment and growth capacity at these sizes. There was no difference between diploid and triploid trout in the mean size of muscle fibres; however, the number of fibres per unit area was reduced by 10% in triploids. No differences were observed in protein or DNA concentrations in muscle tissues between the two genetic groups. Since triploid nuclei have 1·5 times more DNA than diploid nuclei, this deviation from the expected muscle DNA concentration (1·3–1·4 times more DNA in triploids when the 10% reduction in fibre density is considered) suggests that the number of nuclei per muscle fibre is reduced. In both diploids and triploids, mean fibre size increased with body length while fibre density decreased. Similarly, protein concentration in the muscle tissue increased and DNA concentration declined with increasing body length. Protein/DNA ratio was strongly and positively correlated with fibre size. These results demonstrate that changes in DNA and protein concentrations can be used to assess hyperplasia and hypertrophy in muscle tissues. However, the morphometric procedure provides better insight into muscle fibre growth as it enables the direct visualization and analysis of muscle fibre distribution patterns.     

Regional variations in fibre growth dynamics of myotomal and caudal fin muscles in relation to body size of a freshwater teleost, Barbus sarana (Cuv. & Val.)

The growth of red fibres in anterior and middle myotomal regions of B. sarana was mainly by hyperplasia in smaller size classes. In higher size classes, growth by hyperplasia was greater in posterior myotomal region compared to the other two myotomal regions. The growth of pink fibres in anterior myotomal regions was mainly by hypertrophy. The middle and posterior myotomal regions showed fibre growth by hyperplasia. The growth dynamics of white fibres revealed more or less similar pattern in all three myotomal regions against the somatic development. White fibres grew by hyperplasia up to 8 cm F.L. size classes and thereafter by hypertrophy. However, in > 12 cm F.L. size classes, the mean diameter of white fibres did not increase significantly. Similar pattern of growth was found in the white fibres of caudal fin muscle. It is interesting to note that the hyperplasia was mostly completed in the white fibres of the smallest fish studies, whereas, it continued to quite larger fish size in red and pink fibres. Thus, hyperplasia and hypertrophy may be responsible for growth in all fibre types in all myotomal regions in relation to somatic development in this small and medium growing species.     

Growth dynamics of white myotomal muscle fibres in the bluntnose minnow, Pimephales notatus Rafinesque, and comparison with rainbow trout, Salmo gairdneri Richardson

The dynamics of increase of the predominant white muscle fibres of the myotomal bulk in bluntnose minnow, Pimephales notatus , ranging from 2.0 to 9.1 cm f.l. have been analysed by examination of modal progression of fibre diameter frequency classes in fish fed to satiation and growing at different rates at 15, 25 or 30°C. Recruitment of new fibres appeared to contribute little to increase in muscle bulk above 4 cm f.l. , and nothing beyond 6 cm. The dominant means of increase was increase in fibre diameter. The limiting fibre diameter seemed to be 120 μ. These dynamics, which result in an approximately 1: 1 ratio between mean fibre diameter and f.l. , are in contrast to those of the myotomal white muscle of rainbow trout, Salmo gairdneri , in which, regardless of differences in somatic growth produced by temperature, ration size or growth hormone administration, mean fibre diameter does not exceed that in bluntnose minnow until trout exceed 30 cm f.l. In trout there is, moreover, input of new fibres up to approximately 50 cm f.l. , when subsequent growth, as in the minnow, is by means of fibre diameter increase. The bluntnose minnow is a small, slow growing species; the rainbow trout is a large, fast growing species. The discussion links these facts with the observed differences in fibre growth dynamics in relation to a hypothesis of interspecific differences in fish growth capability.     

Growth and multiplication of white skeletal muscle fibres in carp larvae in relation to somatic growth rate

A morphometric analysis of white axial muscle of common carp Cyprinus carpio was undertaken in order to quantify increase in fibre size, fibre nuclei and fibre number in relation to somatic growth rate during early life. In fast-growing carp larvae fed zooplankton, length and height of fibres from the central part of dorsolateral muscle increased at the same rate (0.75) relative to the total length of the larvae during the first 2 weeks of feeding. During this period, the number of nuclei per fibre increased threefold while the number of nuclei per unit fibre surface remained constant. In fast-growing larvae fed a formulated diet, the total cross-sectional area of one epaxial quadrant of white muscle and the total area of white fibres increased at almost the same rate (3.15; 3.23) relative to larval total length during the first 28 days of exogenous feeding. The total number of white fibres increased faster (2.07) relative to the total length of larvae than the mean area of white fibres (1.16). Hyperplasia accounted for 64% of muscle growth in these larvae. The proportion of fibres with a width < 10 μm decreased from 72% at first feeding to 14% 28 days later, while the proportion of fibres with a width >20 μm which was 0% at first feeding increased up to 34% in the same time. The recruitment of new white fibres seemed to be almost the same in the whole muscle quadrant at first feeding and 18 or 28 days later but, 8 days after first feeding, a transient significant recruitment of new fibres was shown at the apex of the myotome. Comparisons between fast- and slow-growing groups of larvae showed that for a given larval total length: (1) the mean width of central white fibres was higher and the proportion of central fibres with a width <10 μm was lower in slow-growing larvae than in fast-growing ones; (2) the total number of white fibres was lower for a higher total cross-sectional area of white muscle in slow-growing larvae than in fast-growing ones. These results suggest that, in Cyprinus carpio larvae, slow-growing conditions are related to a decreased contribution of hyperplasia to muscle growth.     

Post-larval growth in the lateral white muscle of the eel, Anguilla anguilla

The post-larval growth of lateral white muscle was studied in eels at different stages of post-larval development (glass, yellow and silver eels) by means of histochemical methods for myosin-ATPase (mATPase) activity, immunohistochemistry (for myosin isoforms) and electron microscopy.
Morphological, histo- and immunohistochemical data reveal a uniform appearance of white muscle in glass eels, whereas in following stages the typical mosaic appearance is present. Small-diameter fibres show a more acid-labile mATPase activity than large fibres and react with anti-F, anti-FHC and anti-S sera, but not with anti-SHC serum. In the silver stage, the small fibres tend to decrease in number as the size of the eels increases.
Electron microscopy reveals the presence of satellite cells at every stage: in glass eels there are also 'activated' elements showing scarce myofilaments in their cytoplasm; in yellow eels very small fibres are present, enveloped within the basal lamina of well-differentiated muscle fibres; in silver eels there are no fibres showing signs of immaturity.
Presumably the post-larval development of white muscle involves in juvenile eels a substantial recruitment of fibres from the satellite cell population; later the hyperplasia decreases or ceases and hypertrophy remains the only mechanism for muscle growth.     

Uptake of tritiated thymidine in muscle of juvenile carp

In juvenile carp (4.5–6 cm s.l .) labelled myosatellite cell nuclei are found 24 h after injection of tritiated thymidine, but labelled myonuclei after 48 h, indicating that fish myonuclei do not proliferate but originate from undifferentiated myogenic cells. The time pattern accords with the estimated cell-cycle time of adult myogenic cells of carp.     

Influence of growth rate on white muscle dynamics in rainbow trout and brook trout

In trout, fast growth stimulated white muscle fibre hypertrophy ( P 0·001) and hyperplasia ( P <0·01) in outer fibres but not in deep fibres. Glycogen was most prevalent in outer fibres ( P <0·01) and in brook trout ( P <0·01) that on average had three to four times larger fibres than rainbow trout.     

Sexual differences in the increase of white muscle fibres in Argentine hake, Merluccius hubbsi, from the San Matias Gulf (Argentina)

Growth dynamics of white fibres from axial muscle has been investigated in Argentine hake, Merluccius hubbsi , discriminating between sexes for the first time. The frequency distributions of fibre diameters are remarkably different in both sexes at sizes between 42 and 63·9 cm total length (T.l.). Males have a much lower proportion of newly recruited fibres (0–10 μm) than females; at 52–53·9 cm T.l., females have 4% of fibres in that category and males 0·5%. It appears that, from this size interval onwards, the increase in muscle mass is due only to the increase in diameter of individual fibres, which may exceed 300 μm. The lower recruitment rate of new fibres in males, and its relationship to lower growth rates and smaller final sizes, are discussed, and possible effects of reproductive activity are considered.     

Temperature- and developmentally-induced variation in the histochemical profile of myofibrillar ATPase activity in carp

The histochemical profile of calcium activated acid stable myofibrillar ATPase (mATPase) activity in developing larval and juvenile carp was investigated. In the larval fish, differentiation of pink muscle fibres occurred after metamorphosis which was delayed by a week at 17° C compared to larvae grown at 27° C. After metamorphosis the 27° C group exhibited some small myofibres with acid stable mATPase activity in the deep white muscle. This was similar for the juvenile carp which were acclimated for more than a month at 25° C. In contrast, the cold (12° C) acclimated juvenile fish, contained very few small white muscle fibres with acid stable mATPase activity. It was also noted that the cold acclimated fish had lower background acid stable mATPase activity than the warm acclimated fish. Results indicate that after metamorphosis and more evidently in juveniles, temperature can influence the rate of myofibre hyperplasia.     

Sustained swimming speeds and myotomal muscle function in the trout, Salmo gairdneri

Rainbow trout were trained for 3–4 weeks in a flume at swimming speeds of 1, 2 and 3 l s⁻¹. For each experiment growth rates were estimated and by measuring the hypertrophy of red and mosaic skeletal muscle fibres their function was described at particular swimming speeds and compared with earlier experiments on coalfish using the same technique.
Maximum growth, compared with controls in still water, occurred at swimming speeds of 1 l s⁻¹. At this speed the trout mosaic muscle fibres hypertrophied by 40% but the red muscle fibres showed only a 25% hypertrophy. It is suggested that natural swimming speeds are close to 1Ls^−l and the trout mosaic fibres are better adapted for use at this speed in comparison with coalfish white muscle fibres.     

Control of skeletal muscle fibres and adipose cells size in the flesh of rainbow trout

The distributions of the diameters of skeletal muscle fibres and adipocytes were studied in rainbow trout. The cellularity of perivisceral adipose tissues and subcutaneous ventral and dorsal adipose tissues were characterized more specifically. In these tissues, a population of small adipocytes was distinguishable from larger adipocytes. The same was observed in white muscle. The effects of extrinsic factors (dietary lipid in two different thermal conditions) and intrinsic factors (strains in two different saline conditions, growth hormone) on the long-term response of the cellularity of both muscle and adipose tissues were studied. The effects of thermal environment were tested on fish fed the same ration and the effects of saline environment on fish fed ad libitum. The mean size of white muscle fibres was relatively unaffected by the different treatments tested: genetic origin and dietary lipid in different environmental conditions. There were significant differences in growth rate due to genetic origin and saline environment. The possible involvement of hyperplasia in response to these different factors is discussed. Growth hormone supplementation enhanced the percentage of small diameter fibres indicating a role of this hormone in the control of muscle hyperplastic growth. The mean size of adipose cells was affected only slightly by the different treatments tested. An increase in adipose cell size with aging and lipid content was observed. The percentage of small adipocytes also increased with aging. Thus, it is proposed that the development of adipose tissues, and thus fat retention, both result from the recruitment of new adipocytes and from the increase in size of existing adipocytes. The hyperplastic process contributed significantly to the differences in fat retention due to different treatments tested (strains, thermal and saline environments). When partially substituting fish oils for corn oils in the diet, a large increase in the ventral adipose cell size was seen indicating a potential negative effect of n-6 fatty acids on cell proliferation. Growth hormone treatment, on the contrary, induced a decrease in the size of perivisceral adipocytes. Thus, diet and hormonal status affect adipose cells size through two different metabolic pathways: lipogenesis and lipolysis respectively.     

Posthatch development of the axial musculature of the common dentex Dentex dentex, L (Teleostei)

The common dentex is a promising candidate for Mediterranean aquaculture. The present work is aimed at describing the development of the axial musculature from hatching to postlarval life. Transmission electron microscopy, histochemical (NADH-TR and mATPase) and immunohistochemical techniques (S-58 and TUNEL) have been used. At hatching superficial red and deep white muscles can be distinguished. Presumptive dermomyotome (external) cells are initially located over the superficial red muscle but shortly (2 days) tend to concentrate towards the epaxial and hipaxial limits of the myotome. Then, these cells enter the myotome and spread around and within the white muscle thus being apparently responsible for the stratified hyperplasia of the myotome. Mosaic hyperplasia is activated during the second half of the larval period and initially relies on differentiation of a population of atypical premyoblastic cells (APC). APC are mononuclear cells with euchromatic nuclei, cytoplasms full of thin longitudinally projected tubules, occasional mitochondria and scattered ribosomes. By the end of the larval period these cells tend to disappear, partly due to apoptosis, but postlarval mosaic hyperplasia continues by differentiation of presumptive myosatellite cells. APC are an unexpected and singular finding of this study which deserves more research, so as to further characterize their ancestry, developmental programme and fate. In addition to the white and superficial red muscle fibres, intermediate (pink) and tonic fibres appear during larval metamorphosis. Later, during the early postlarval life, a new type of slow twitch red muscle fibre is differentiated (red adult type).     

Growth dynamics of white and red muscle fibres in fast- and slow-growing strains of rainbow trout

Compared with fish of a slow-growing strain, fast-growing rainbow trout exhibited significantly smaller white fibre diameters, throughout development from hatching to 24 cm body length, although possessing similar total number of fibres. In contrast, in red muscle, no differences were observed in fibre diameter between the two strains, but the fast growing fish showed a significantly higher number of red fibres. The differences in growth rate between the two strains were related to the mean white fibre diameter and were found to be matched by proportional adjustments in recruitment of new fibres to the growing muscle. Thus, the largest and fastestgrowing strain showed evidence of sustained higher recruitment of muscle fibres that endowed this strain with the potential to maintain rapid somatic growth for longer and accomplish greater muscle growth.     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish